The ability to control aspects of polyketide biosynthesis has stimulated interest in the combinatorial engineering of novel antibiotics. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. For example, there has been interest in making polyketides in host cells that are otherwise unable to make polyketides. E. coli host cells, among other host cells which do not otherwise make polyketides, have been contemplated that incorporate expressions systems for expressing polyketides synthases to make polyketides. See PCT publication No. 01/27306 which is incorporated herein by reference.
Polyketide biosynthesis involves the condensation of malonyl CoA, methylmalonyl CoA, or related substituted malonyl CoA precursors, also known as “extender units,” onto an acetyl CoA, propionyl CoA, or related acyl CoA precursor, also known as a “starter unit.” Of the myriad host cells possible for utilization as polyketide producing hosts, many do not naturally produce such substrates (i.e., polyketide precursors) or do not produce the substrates (i.e., polyketide precursors) in adequate amounts. For example, E. coli does not produce methylmalonyl CoA in sufficient quantities for polyketide synthesis using methylmalonyl CoA as a substrate (precursor). The introduction and modification of biochemical pathways for methylmalonyl CoA biosynthesis in host cells that do not otherwise make polyketides have also been contemplated. See PCT publication Nos. 01/27306 and 01/31035, which are incorporated herein by reference. Two biochemical pathways involving methylmalonyl CoA are relevant to the present invention as well. These pathways are the methylmalonyl CoA mutase pathway, hereafter referred to as the MUT pathway, and the propionyl CoA carboxylase pathway, hereafter referred to as the PCC pathway. See PCT/US 02/06399.
Although approaches to make polyketide in organisms that otherwise do not make polyketides, or make them in low quantities, are extremely useful, it would be useful to produce polyketides that differ from naturally occurring polyketides by incorporating different starter units not found in naturally occurring polyketides.
Interest in making non-natural polyketides has led to organisms that produce polyketides using modified modular polyketide synthase systems wherein directed modification incapacitates the system from using its natural starting material. Novel polyketides have been synthesized by overriding the starter module and supplying a variety of suitable diketide substrates in the form of NAC thioesiers or other suitable thioester. See PCT patent publication Nos. 97/02358 and 99/03986, each of which is incorporated herein by reference. In particular, 15-methyl-6-deoxyerythronolide B (15-methyl-6-dEB) can be made using a 6-dEB synthase (DEBS) gene, by feeding a propyl diketide thioester (e.g., (2S,3R)-3-hydroxy-2 -methylhexanoate N-acetylcysteamine thioester) to DEBS. The resulting polyketide, 15-methyl-6-dEB, is also referred to as 13-propyl-6-dEB because it has a propyl group at the 13-position replacing the ethyl group found in 6-dEB. While the diketide feeding technology provides useful amounts of compound, the cost of producing polyketides by that technology is increased by the need to prepare the synthetic diketide. Moreover, certain polyketide producing cells degrade some of the diketide before it can be incorporated into a polyketide by the PKS, thus increasing the cost of production. Methods to produce polyketides by other means could be more efficient and cost effective, therefore, if the need to feed costly synthetic diketide substrates to the PKS were eliminated, in order to produce non-naturally occurring polyketides.
Given the potential for making valuable and useful novel polyketides in large quantities in heterologous host cells, there is a need for host cells capable of making the substrates (i.e., precursors) required for novel polyketide biosynthesis. The present invention helps to meet this need by providing recombinant host cells that contain a PKS expression system, and that also contain one or more expression systems for making substrates (i.e., precursors) for novel polyketide biosynthesis.